Brain cancer is the second leading cause of cancer-related deaths in children in the United States, with approximately 5000 new cases each year (Cancer Facts & Figures 2015, Atlanta: American Cancer Society). More specifically, medulloblastoma is the most common malignant brain tumor in children, accounting for 25-30% of primary central nervous system (CNS) tumors (Cancer Facts & Figures 2015, Atlanta: American Cancer Society). Current available treatment protocols for medulloblastomas include surgical removal of the tumor, fractionated radiation therapy (RT) and intensive chemotherapy treatments (Adamski et al., F1000Prime Rep., 2014;6: 56). Although, such an intense treatment regimen yields a promising average 5-year survival rate of 60-80% (Gajjar and Robinson, Nat. Rev. Clin. Oncol., 2014, 11: 714-722), nearly all survivors experience hindered quality of life and long-term cognitive dysfunction due to aggressive RT (Castelo-Branco et al., Pediatr Res., 2012, 71: 523-528). Moreover, one of the most problematic issues stems from the recurrence of the disease which occurs in almost 20% of patients post-treatment, resulting in a 5-year survival rate of less than 5% (Zeltzer et al., J Clin Oncol., 1999, 17: 832-845).
Medulloblastoma is classified as a stage IV primary tumor that originates in the cerebellum (Polkinghorn and Tarbell, Nat. Clin. Pract. Oncol., 2007, 4:295-304). Due to its wide variability in molecular, histological and clinical profiles, medulloblastoma has been classified into 4 unique profiles based on transcriptional studies (Taylor et al., Acta Neuropathol., 2012, 123:465-472). In 2012, an international consensus was reached in naming the four groups, Sonic hedgehog (Shh), Wingless (Wnt), Group C and Group D (Taylor et al., Acta Neuropathol., 2012, 123:465-472). The Shh and Wnt groups are named according to the regulating pathways that cause this specific subtype, while the specific driver mutations for groups C and D remain unknown. Both the Wnt and Shh pathways have been implicated in promoting the development of medulloblastoma during embryogenesis, and targeting these specific signaling molecules have been shown to reduce tumor growth in vivo (Brun et al., Oncogene, 2015, doi:10.1038/onc.2014.304, Matsuo et al., Toxicol. Pathol., 2014, 42:1174-1187, Baryawno et al., Cancer Res., 2010, 70:266-276).
Regardless of the specific signaling pathway involved in medulloblastomas, the underlying cause of this type of cancer seems to arise from the dysregulation of normal stem/progenitor cells during development (Polkinghorn and Tarbell, Nat. Clin. Pract. Oncol., 2007, 4:295-304). In essence, when normal stem cells lose their homeostatic proliferative functions, they develop a mutated phenotype and transform into cancer stem cells (CSCs) (Hope et al., Nat. Immunol., 2004, 5:738-743) which lead to the tumor initiation and progression of medulloblastomas (Manoranjan et al., Pediatr. Res., 2012, 71:516-522).
Cancer stem cells (CSCs) are a subpopulation of cancer cells within tumors with the ability to perpetually self-renew and differentiate, providing tumors with a limitless supply of cancer cells (Jung et al., Arch. Pharm. Res., 2015, 38:414-422). To date, available conventional treatment therapies are only efficient in targeting the tumor bulk and fail to eradicate the tumor-initiating CSCs residing within the bulk. Such therapies allow for the survival of CSCs post-treatment and result in the repopulation of tumors and relapse of the disease. However, if CSCs can be specifically targeted and eradicated with treatment, then tumor recurrence can be eliminated.
Therefore, it is important to develop cancer therapeutics capable of identifying and targeting CSCs to prevent cancer relapse. To date, there are very few drugs available that are capable of distinguishing CSCs from normal stem cells (Chen et al., Acta Pharmacol. Sin., 2013, 34:732-740). In recent years, several laboratories have identified novel cell-surface markers specific to CSCs in breast, colon, brain and prostate cancers (Hu et al., Am. J. Cancer Res., 2012, 2:340-356). More specifically, CD133 (prominin-1), CD44 (cluster of differentiation 44) and LGR5 (leucine-rich repeat containing G protein-coupled receptor 5) CSC markers have all been shown to be associated with tumorigenesis and stemness potential of medulloblastoma (Blazek et al., Int. J. Radiat. Oncol. Biol. Phys., 2007, 67:1-5, Whittier et al., Acta Neuropathol. Commun., 2013, 1:66, Parker et al., Anticancer Res., 2005, 25:3855-3863).
For years, CSCs have been shown to have a unique ability to resist radiation exposure, minimize inflicted radiation-induced damage and evade apoptosis (Rycaj et al., Int. J. Radiat. Biol., 2014, 90:615-621). The radio-resistant property of CSCs is believed to be due to their ability to activate a more rapid DNA repair mechanism compared to normal stem cells, allowing them to escape cellular death (Rycaj et al., Int. J. Radiat. Biol., 2014, 90:615-621). Current brain cancer therapies, such as x-ray and gamma ray ionizing radiation, result in both single stranded and double stranded DNA breaks (Aparicio et al., DNA Repair (Amst), 2014, 19:169-175). Single-stranded breaks (SSBs) are well-tolerated by the cells because the strand is able to ligate and repair itself rapidly. On the other hand, double-stranded breaks (DSBs) are the most biologically lethal type of DNA damage and a major focus for developing novel radiotherapy cancer strategies (Aparicio et al., DNA Repair (Amst), 2014, 19:169-175). Upon radiation-induced DSBs, a DNA damage response signaling cascade is activated and triggers a chain of events which include: 1) identification of the damage, 2) arrest of the cell cycle and 3) DNA repair via non-homologous end joining or homologous recombination (Raleigh and Haas-Kogan, Future Oncol., 2013, 9:219-233). In some cases, cells are damaged to an irreparable extent and are thus prohibited from exiting the cell cycle arrest (Raleigh and Haas-Kogan, Future Oncol., 2013, 9:219-233). Instead, they either undergo apoptosis or initiate cell senescence (Raleigh and Haas-Kogan, Future Oncol., 2013, 9:219-233). However, in the event cells can be repaired, selection and activation of the appropriate repair pathway is dependent on the phase at which the cells were cycling through at the time of the damage.
The NF-κB pathway is a widely studied signaling pathway that is involved in various processes, including inflammation and cancer (Hoesel and Schmid, Mol. Cancer., 2013, 12:86). In normal conditions the NF-κB pathway is tightly regulated and is only activated in response to specific cellular signals (Hoesel and Schmid, Mol. Cancer., 2013, 12:86). When inactive, NF-κB is bound to an inhibitory IκBα complex and remains sequestered in the cytoplasm. Upon cellular stimulus, a signaling cascade is activated in which IκBα (inhibitor of KB) is phosphorylated by IKK (IkB kinase). Phosphorylation of IκBα results in the release of IκBα from NF-κB, thereby activating NF-κB and promoting its translocation into the nucleus. NF-κB then binds to DNA and activates the transcription of various pro-survival genes. In contrast, in CSCs, the NF-κB signaling pathway is deregulated and constitutively active (Hoesel and Schmid, Mol. Cancer., 2013, 12:86). This allows the cells to over proliferate and avoid apoptosis, thereby increasing their radio-resistance and tumorigenic potential (Karin et al., Nat. Rev. Cancer., 2002, 2:301-310, Spiller et al., BMC Cancer. 2011, 11:136). Several laboratories are trying to develop novel NF-κB inhibitors in an attempt to block the transcription of these pro-tumorigenic genes (Pal et al., J. Inflamm. (Lond)., 2014, 11:23, Fuchs et al., Curr. Mol. Pharmacol., 2010, 3: 98-122, Sharma et al., Curr. Med. Chem., 2007, 14:1061-1074).
Thrombin peptide TP508, also known as rusalatide acetate or Chrysalin®, is a 23-amino acid synthetic peptide representing a portion of the human prothrombin with a sequence of AGYKPDEGKRGDACEGDSGGPFV (SEQ ID NO:6). TP508 corresponds to amino acids 508-530 of the prothrombin or 183-200 of the α-thrombin peptide. Thrombin plays an important role in the coagulation cascade by converting soluble fibrinogen into the insoluble fibrin required for blood clot formation (Krishnaswamy, J. Thromb. Haemost., 2013, 11 Suppl 1:265-276). Upon vascular injury, prothrombin is proteolytically cleaved by activated factor X, yielding the biologically active α-thrombin (Krishnaswamy, J. Thromb. Haemost., 2013, 11 Suppl 1:265-276). The biological activity of the TP508 sequence was discovered by screening molecules that could bind to high-affinity thrombin receptors and mimic cellular effects of thrombin at sites of tissue injury (Carney et al., Semin. Thromb. Hemost., 1986, 12:231-240, Glenn et al., Pept. Res., 1988, 1:65-73). Thus, TP508 was selected for its interaction with a subset of high affinity non-proteolytic thrombin receptors (NPARs). Early studies demonstrated specificity of TP508 binding and crosslinking to the NPAR receptor, specific signaling cascades that included activation of endothelial nitric oxide synthase, PI3K, SRC, AKT and PKC.